Although intestinal intraepithelial lymphocytes (IEL) constitute one of the largest T cell compartments of the body our understanding of their development and function is conspicuously limited. The objective of the presented study is to provide insight into the most fundamental aspects of IEL biology, addressing the question of their antigen-major histocompatibility complex (MHC) restriction and potentially also their antigen specificity. Our research will focus on CD8aa TCRa[unreadable] IEL populations. In the first part of this project, we will immortalize IEL by fusing them with a thymoma cell line to generate T-cell hybridomas that will be subsequently used in a number of functional assays. Initially, they will be stimulated with different antigen preparations (such as intraepithelial cell lysates, bacterial lysates, food antigen preparations) presented by a variety of cells including various populations of dendritic cells and epithelial cells. In case of positive activation, the identification of the restricting MHC molecules will be investigated using antigen-presenting cells isolated from various MHC deficient mice strains. Antigen identification will then be attempted by HPLC sequential fractionations and mass spectrometry. In the second aim, we will sequence and clone the TCRa- and -[unreadable] chains of the hybridomas generated previously. We will then retrovirally transfect these TCRs into bone marrow cells (BM) and inject them into various lethally irradiated MHC hosts. Analysis of the different BM chimeras should allow us to define the MHC restriction of CD8aa TCRa[unreadable] IELs in vivo. Both aims are complementary and will lead to the identification of the antigen and MHC restriction of CD8aa TCRa[unreadable] IELs. The results of this study will provide us with a tremendous amount of new and important information regarding the biology of CD8aa TCRa[unreadable] IELs. Better understanding of the intestinal immune system holds promise of the development of novel therapeutic modalities for a number of intestinal inflammatory disorders. PUBLIC HEALTH RELEVANCE: Given the profound role intestinal immune system plays in generation of tolerance and protective immunity, it is surprising how little the potential of this system has been utilized in clinical practice. Unconventional intraepithelial lymphocytes constitute a substantial population of T cells in the intestine, yet their role remains elusive. Here we set out to explain what are the target antigens for these cells and the mechanisms of their recognition, information that is critical in understanding how the intestinal immune lymphocytes are capable of eliciting an immune response against pathogens.